This application is a 371 of PCT/EP98/05714, filed Sep. 2, 1998, which claims priority from GB 9718901.3, filed Sep. 5, 1997.
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Induction of cytotoxic lymphocyte (CTL) responses occurs naturally during infection of a target cell, or uncontrolled synthesis of a tumor antigen, wherein enzymatic degradation of the target antigen takes place in the cell cytoplasm. This phenomenon allows cytoplasmic peptides derived from the pathogen, or tumor specific antigen, to enter the Th1 (endogenous antigen processing) pathway and be presented on the surface of the cell associated with an MHC class 1 molecule. If a vaccine antigen does not enter into the cytoplasm of the host cell, then it might be taken up by the cell and enter the exogenous antigen processing pathway and ultimately be presented on the surface of the cell associated with a MHC class II molecule. This alternative route generally results in T-helper responses and antigen specific antibody responses.
After conventional vaccination with subunit or non-living vaccines, an antigen generally does not enter the cytoplasm of a host cell, and therefore will not enter the endogenous antigen processing pathway and ultimately will not induce a CTL response. CTL induction is believed to correlate with Th-1 cytokine profile responses, specifically with IFN-xcex3 and IL-2 secretion. IFN-xcex3 secretion is associated with protective responses against intracellular pathogens, including parasites, bacteria and viruses. Activation of leucocytes by IFN-xcex3 enhances killing of intracellular pathogens and increases expression of Fc receptors. Direct cytotoxicity may also occur, especially in synergy with lymphotoxin (another product of TH1 cells). IFN-xcex3 is also both an inducer and a product of NK cells, which are major innate effectors of protection. TH1 type responses, either through IFN-xcex3 or other mechanisms, provide preferential help for murine IgG2a, and human IgG1, immunoglobulin isotypes.
International patent application No. WO 95/17210 discloses an adjuvant emulsion system based on squalene, xcex1-tocopherol, and polyoxyethylene sorbitan monooleate (TWEEN 80), optionally formulated with the immunostimulants QS21 and/or 3D-MPL. This adjuvant formulation is a very potent inducer of a wide range of immune responses.
These oil in water emulsions, when formulated with 3 De-O-acylated monophosphoryl lipid A (3D-MPL) and QS21 are potent inducers of Th1 type immune responses. Accordingly, this system when associated with antigen preferentially stimulate the sub-isotype of IgG associated with Th1 responses (for example, murine IgG2a and human IgG1) and also will induce significant levels of IFN-xcex3 production and antigen specific CTL responses. The observation that the basic oil in water/QS21/3D-MPL formulation can induce strong CTL responses is significant, as these responses in certain animal models have been shown to induce protection against disease.
Immunologically active saponin fractions (e.g. Quil A) having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina are known in the art. Derivatives of Quil A, for example QS21 (an HPLC purified fraction derivative of Quil A), and the method of its production is disclosed in U.S. Pat. No. 5,057,540. Amongst QS21 (known as QA21) other fractions such as QA 17 are also disclosed. The use of such saponins in isolation is accompanied with disadvantage in that local necrosis, that is to say, localized tissue death, occurs at the injection site, thereby leading to pain.
3 De-O-acylated monophosphoryl lipid A is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3 De-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be prepared by the methods taught in GB 2122204B. A preferred form of 3 De-O-acylated monophosphoryl lipid A is in the form of an emulsion having a small particle size less than 0.2 xcexccm in diameter, and its method of manufacture is disclosed in European Patent No. EP 0 671 948 B1.
In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system has to comprise a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as xe2x80x9cbeing capable of being transformed by metabolismxe2x80x9d (Dorland""s Illustrated Medical Dictionary, W. B. Sanders Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE(copyright) and others. Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention. Squalene is a metabolizable oil virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619).
Oil in water emulsions per se are well known in the art, and have been suggested to be usefull as adjuvant compositions (EPO 399843).
The oil in water emulsions described in International patent application No. WO 95/17210 obviously hold great advantages over conventional non-Th1 inducing adjuvants. However, the inclusion of QS21 has so far made this potent adjuvant reactogenic, leading to pain at the site of injection.
Formulations comprising QS21 with a sterol are known from International Patent Application No. PCT/EP96/01464. No oil in water emulsions are disclosed in this document. Sterols are well known in the art, for example cholesterol is well known and is, for example, disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat.
The present invention relates to an oil in water emulsion compositions, their use in medicine, in particular to their use in augmenting immune responses to a wide range of antigens, and to methods of their manufacture; the oil in water emulsion comprising a metabolizable oil, a saponin and a sterol.